Post-consolidation maintenance therapy with 13-cis retinoic acid (13-cisRA) and the chimeric anti-GD2 antibody dinutuximab (Unituxin) improves the survival of high-risk neuroblastoma (NB) patients. Despite currently available optimal therapy > 40% of children still develop recurrent disease, which is fatal for most. In the current proposal we seek to identify molecular mechanisms of progressive disease in NB patients. Although MYC genomic amplification is seen in 1% of NB patients, c-MYC protein is overexpressed in 11% of patients at diagnosis. Our preliminary data show that high c-MYC protein expression is far more frequent in progressive disease relative to diagnosis using patient-derived cell lines established from clinical samples at progressive disease and at diagnosis. In searching the mechanisms of MYC transcriptional activation, we demonstrated that transcription factors, OCT4 and TCF 3 were elevated in a cell line model selected to express high c-MYC in replicating the clinical treatment schedule of maintenance therapy (13-cisRA) in high-risk neuroblastoma. When OCT4 was knocked-down, the expression of c-MYC was decreased and the sensitivity to 13-cisRA was restored. Subsequently, we identified two kinases, MAPKAPK2 (MK2) and DNAPK, which are predicted to bind and phosphorylate OCT4. The MK2-OCT4-c-MYC axis was confirmed in another progressive disease model of neuroblastoma with high c-MYC expression. Based on our preliminary data, we hypothesize 1) that kinase(s) phosphorylate OCT4 and the phosphorylated OCT4-induces transcriptional activation of c-MYC and that those kinases can be targeted to inhibit the activation of OCT4-induced c-MYC overexpression, and thus the expression of the kinase(s) could be biomarkers and therapeutic targets for patients with progressive disease with high c-MYC. Our goals are: 1) to employ our extensive panel of NB cell lines and patient-derived xenografts (PDXs) to define the specific roles of OCT4 and its phosphorylation regulation by MK2 and DNA-PKcs kinases in a c-MYC overexpression state that causes resistance to one of the maintenance therapy in NB, 2) to validate them as potential markers of poor outcome in collaboration with the Children's Oncology Group (COG) in patient tumor samples, and 3) to demonstrate the feasibility of inhibiting key kinase(s) to modulate OCT4/c-MYC axis to enhance activity of chemotherapy in NB cell lines and patient-derived xenografts (PDXs). The ultimate goal of the proposed study is to validate regulation of c-MYC as a novel mechanism of tumor progression in neuroblastoma and to identify a druggable target(s) for drug-resistant recurrent neuroblastoma.